Pyemotes tritici, the straw-itch mite, is one of thirteen known species of mites in the genus Pyemotes, all of which are predatory and which possess venoms causing mild to extreme toxicity in target insects. The thirteen known species can be divided into two morphological groups which also differ in host range, methods of dispersal and toxicity to their hosts, and in the effects of their toxins on insects and man. The scolyti and ventricosus groups are summarized in Table 1. Most members of the ventricosus group have extremely insect-toxic venoms. The scolyti mites are all phoretic, and are generally found on bark beetles; they may express paralytic toxins.
The mite life cycle takes only 7-14 days, with 100-300 newborn sexually mature mites emerging from the mother. When a female emerges, it immediately mates and finds a new host. The time for paralysis of a host insect is variable, and appears to depend on the species, size, developmental stage and number of attacking mites. All stages of host insects may be attacked by the mites, but adults are generally less susceptible due to their more sclerotized (i.e. harder) cuticles, which are more difficult for the mite mouthparts to penetrate.
The mite venoms themselves do not appear to be specific for particular insects, since the venoms are toxic to a wide variety of insect host and nonhost species. The toxin(s) cause irreversible paralysis without disrupting respiratory mechanisms (Weiser and Slama (1964) Ann. Ent. Soc. Am. 57:479).
Insect-specific toxins in the venom of P. tritici, have been purified and characterized (Tomalski et al. (1988) Toxicon 26:127-132; Tomalski et al. (1989) Toxicon 27:1151-1167). These toxins are produced in female mites and injected into insect prey as components of the venom, resulting in paralysis of the insect prey. The paralysis allows the feeding female mite to become fully gravid, thus ensuring adequate nutrients for reproduction. Low molecular weight toxin components cause rapid contractile muscle paralysis while a high molecular weight toxin fraction causes flaccid muscle paralysis.
One toxin component, designated TxP-I, has been purified to apparent homogeneity; it has an apparent molecular weight of 27,000, as determined by SDS-polyacrylamide gel electrophoresis. An analysis of the amino acid composition of TxP-I was presented in Tomalski et al. (1989), supra. The relatively high cysteine content could result in a number of disulfide bonds in the toxin molecule. The N-terminal sequence of TxP-I has been published: N-asp-asn-gly-asn-val-glu-ser-val-arg-ala-val-val-ile-asp-tyr-[Xaa]-asp-il e-arg-his-pro-(SEQ ID NO:1). The N-terminal amino acid sequence was not found to be homologous to any protein sequence in the Protein Identification Resource (National Biomedical Foundation Release No. 13, Jun. 30, 1987).
Two other components were resolved which exhibit molecular weights of 28,000 and 29,000; these two components comprise TxP-II. Based on peptide mapping and immunoblot experiments, it was postulated that the protein components of TxP-I and TxP-II are isoproteins (Tomalski et al. (1989) supra). The mixture of TxP-I and TxP-II comprise TxP-III.
Preparations of P. tritici toxins are not acutely toxic to mammals, as tested with mice by either intraperitoneal or intracerebral routes. The doses which cause paralysis of 50% of the test insects (PD.sub.50) for TxP-I, TxP-II and TxP-III are 330, 550 and 500 micrograms/kg, respectively when tested with wax moth (Galleria mellonella) larvae. Txp-I and Txp-II cause rapid muscle-contracting paralysis.
Polyclonal antibody has been produced using purified TxP-I as the antigen. This antibody was reactive against both TxP-I and Txp-II, and the antibody neutralized the paralytic activity of partially purified preparations of TxP-III. (Tomalski et al. (1989) supra).
Insect-specific proteinaceous neurotoxins have been found in the venoms of other arthropods including scorpions, wasps and spiders (Zlotkin (1985) in Comprehensive Insect Physiology, Biochemistry and Pharmacology. I. Insects. I. Kerkut and L. I. Gilbert (eds.) Pergamon Press, Oxford, U.K., pp. 499-546. Several of the peptide toxins from scorpions exhibit insect-specific neurotoxic effects and have been sequenced. These scorpion toxins are of relatively low molecular weight, i.e. from about 3000 to about 8000 daltons, considerably different from the mite toxins. There is no apparent sequence relationship between the mite and scorpion toxins, but both mite and scorpion toxins have high cysteine content. Compact toxin protein structures are stabilized by disulfide bonds.
Interest in the biological control of insect pests has arisen as a result of disadvantages of conventional chemical pesticides. Chemical pesticides generally affect beneficial as well as nonbeneficial species. Insect pests tend to acquire resistance to such chemicals so that new insect pest populations can rapidly develop that are resistant to these pesticides. Furthermore, chemical residues pose environmental hazards and possible health concerns. Biological control presents an alternative means of pest control which can reduce dependence on chemical pesticides.
Strategies for biological control include the deployment of naturally-occurring organisms which are pathogenic to insects (entomopathogens) and the development of crops that are more resistant to insect pests. Approaches include the identification and characterization of insect genes or gene products which may serve as suitable targets for insect control agents, the identification and exploitation of previously unused microorganisms (including the modification of naturally-occurring nonpathogenic microorganisms to render them pathogenic to insects), the modification and refinement of currently used entomopathogens, and the development of genetically engineered crops which display greater resistance to insect pests.
Viruses that cause natural epizoptic diseases within specific insect populations are among the entomopathogens which have been developed as biological pesticides. Entomopathogenic viruses include the baculoviruses, entomopoxviruses, reoviridae (cytoplasmic polyhedrosis viruses), iridoviruses, parvoviruses, rhabdoviruses, picornaviruses, nodaviruses, ascoviruses (still unclassified) and probably certain retroviruses.
Baculoviruses are a large group of evolutionarily related viruses which infect only arthropods (Miller, L. K. (1981) in Genetic Engineering in the Plant Sciences. N. Panopoulous, (ed.), Praeger publ., New York, pp. 203-224; Carstens, (1980) Trends in Biochemical Science 52:107-110; Harrap and Payne (1979) in Advances in Virus Research. Vol. 25, Lawfer et al. (eds.), Academic Press, New York, pp. 273-355, Granados, R. R. and Federici, B. A. eds. (1986) The Biology of Baculoviruses, Volume 1, Biological Properties and Molecular Biology CRC Press Inc., Boca Raton, Florida). Some baculoviruses only infect insects which are pests of commercially important agricultural and forestry crops. Other baculoviruses are known which specifically infect other insect pests, e.g., mosquitoes and fleas. Such baculoviruses are potentially valuable as biological control agents. A potential advantage of baculoviruses as biological pesticides is their host specificity. Baculoviruses as a group infect only arthropods, and individual baculovirus strains usually only infect one or a few species of insects. Thus, they pose little or no risk to man or the environment, and can be used without adversely affecting beneficial insect species.
Baculovirus subgroups include nuclear polyhedrosis viruses (NPV), granulosis viruses (GV), and nonoccluded baculoviruses. In the occluded forms of baculoviruses, the virions (enveloped nucleocapsids) are embedded in a crystalline protein matrix. This structure, referred to as an inclusion or occlusion body, is the form found extraorganismally in nature and is responsible for spreading the infection between organisms. The characteristic feature of the NPV group is that many virions are embedded in each occlusion body, which is relatively large (up to 5 micrometers). Occlusion bodies of the GV group are smaller and contain a single virion each. The crystalline protein matrix of the occlusion bodies of both forms is primarily composed of a single 25,000 to 33,000 dalton polypeptide which is known as polyhedrin or granulin. Nonoccluded baculoviruses do not produce a polyhedrin protein, and do not form occlusion bodies. Groner et al. in The Biology of Baculoviruses, Volume 1, p, which is incorporated by reference herein, in Chapter 9, Tables 2 and 7 provides an extensive list of NPV hosts and GV hosts, for example.
In nature, infection is initiated when an insect ingests food contaminated with baculovirus particles, typically in the form of occlusion bodies. The occlusion bodies dissociate under the alkaline conditions of the insect midgut, releasing the virions which then invade epithelial cells lining the gut. Within a host cell, the baculovirus migrates to the nucleus where replication takes place. Initially, specific viral proteins are produced within the infected cell via the transcription and translation of so-called "early genes." Among other functions, these proteins are required for the replication of the viral DNA, which begins 4 to 6 hours after the virus enters the cell. Viral DNA replication proceeds up to about 24 hours post-infection (pi). From about 8 to 24 hours pi, infected cells express "late genes" at high levels. These include components of the nucleocapsid which surround the viral DNA during the formation of progeny virus particles. Production of progeny virus particles begins around 12 hours pi. Initially, progeny virus migrate to the cell membrane where they acquire an envelope as they bud out from the surface of the cell. The nonoccluded virus particles can then infect other cells within the insect. Polyhedrin synthesis begins approximately 18 hours after infection and increases to very high levels by 24 to 48 hours pi. At about 24 hrs pi, there is a decrease in the rate of nonoccluded virus production, and most progeny virus particles are then embedded in occlusion bodies. Occlusion body formation continues until the cell dies or lyses. Some baculoviruses infect virtually every tissue in the host insect so that at the end of the infection process, the entire insect is liquified, releasing extremely large numbers of occlusion bodies which can then spread the infection to other insects. (Reviewed in The Biology of Baculoviruses, Vol. I and II, Granados and Federici (eds.), CRC Press, Boca Raton, Florida, 1986.)
Baculoviruses which are derivatives of AcMNPV which are useful as expression vectors have been described in U.S. patent application Ser. No. 07/353,847, filed May 17, 1989 International Patent Application PCT/US90/02814, filed May 17, 1990; Rankin et al. (1988) Gene 70:39-49; Ooi et al. (1989) J. Mol. Biol. 210:721-736, Thiem and Miller (1990) Gene 91:87-95, all of which are incorporated by reference herein. Particularly strong late and very late promoters are described and include the modified polyhedrin promoter LSXIV, the hybrid Cap/Polh promoter and the synthetic promoter Syn.
Baculoviruses which exhibit improved insecticidal properties have been described. For example, AcMNPV in which the egt (ecdysone glucosyl transferase) gene has been inactivated causes earlier cessation of feeding and earlier larvae death as compared to larvae infected with wild-type AcMNPV (O'Reilly and Miller (1989) Science 245:1110-1112; O'Reilly and Miller (1990) J. Virol. 64:1321-1328; U.S. Pat. No. 5,180,580.
Egt.sup.- AcMNPV which have been further genetically altered to express a protein affecting ecdysis can provide additional improvements in insecticidal properties (International Patent Application PCT/US90/03758, filed Jun. 29, 1990, which is incorporated by reference herein). Egt.sup.- AcMNPV derivatives which express juvenile hormone esterase, eclosion hormone, or prothoracicotropic hormone have been constructed. Feeding times of infected larvae were reduced and death occurred earlier than in larvae infected with wild-type or Egt.sup.- AcMNPV.
Maeda (1989) Biochem. Biophys. Res. Commun. 165:1177-1183, has also described a genetically engineered baculovirus with improved pesticidal properties. BmNPV, which infects the silkworm Bombyx mori, has been modified to express a synthetic gene encoding the diuretic hormone of the tobacco hornworm Manduca sexta. The fluid balance of infected insects was disrupted, and killing was about 20% faster than with the wild-type virus.
Dee and co-Workers 1990) Bio/Technology 8:339-342, have cloned and expressed the insecticidal toxin from the scorpion Androctonus australis in mouse fibroblast cells. The coding sequence was fused to the signal peptide sequence of human interleukin-2 and synthesis was directed by promoter sequences in the long terminal repeat of Moloney murine sarcoma virus. The recombinant protein, which was secreted into the extracellular medium, was reported to be toxic to mosquito larvae but not to mouse cells in culture or to mice.
A gene encoding an insect toxin from Buthus eupeus (middle-Asian subspecies of scorpion) has been synthesized, cloned into the genome of AcMNPV (a nuclear polyhedrosis virus from Autographa californica) and expressed under polyhedrin promoter control. Constructions were also made in which the scorpion toxin was expressed from a synthetic gene comprising the toxin coding sequence fused to a signal-peptide coding sequence or as a fusion protein with 58 amino acids of polyhedrin at the N-terminus. In all cases there was some expression as determined by [.sup.35 S]-methionine radiolabeling SDS-polyacrylamide gel electrophoresis and autoradiography, but there was no insect-paralytic activity observed for any of the expression products. It was believed that this was in part due to protein instability, but the failure to detect biological activity may have been the result of insufficient sensitivity in the assay system or due to failure of the recombinant protein to form a functional three-dimensional structure (Carbonell et al. (1988) Gene 73:408-418).
Hammock et al. (1990) Nature 344:458-461 describes the baculovirus-mediated expression of an insect gene encoding juvenile hormone esterase (JHE), an enzyme which inactivates a developmental hormone.
Merryweather et al. (1990) J. Gen. Virology 71:1535-1544 reports the construction of baculovirus containing the Bacillus thurinoiensis subsp. kurstaki HD-73 delta endotoxin. The BTk HD-73 endotoxin gene is placed under the control of the polyhedrin promoter.